Androgenic steroids induce pathologic scarring in a preclinical porcine model via dysfunctional extracellular matrix deposition.

Hypertrophic scarring is a major source of morbidity. Sex hormones are not classically considered modulators of scarring. However, based on increased frequency of hypertrophic scarring in patients on testosterone, we hypothesized that androgenic steroids induce abnormal scarring and developed a preclinical porcine model to explore these effects. Mini-swine underwent castration, received no testosterone (noT) or biweekly testosterone therapy (+T), and underwent excisional wounding. To create a delayed wound healing model, a subset of wounds were re-excised at 2 weeks. Scars from postoperative day 42 (POD42) and delayed wounds (POD28) were harvested 6 weeks after initial wounding for analysis via histology, bulk RNA-seq, and mechanical testing. Histologic analysis of scars from +T animals showed increased mean fibrosis area (16 mm2noT, 28 mm2+T; p = .007) and thickness (0.246 mm2noT, 0.406 mm2+T; p < .001) compared to noT. XX+T and XY+T scars had greater tensile burst strength (p = .024 and p = .013, respectively) compared to noT swine. Color deconvolution analysis revealed greater deposition of type I and type III collagen as well as increased collagen type I:III ratio in +T scars. Dermatopathologist histology scoring showed that +T exposure was associated with worse overall scarring (p < .05). Gene ontology analysis found that testosterone exposure was associated with upregulation of cellular metabolism and immune response gene sets, while testosterone upregulated pathways related to keratinization and laminin formation on pathway analysis. In conclusion, we developed a preclinical porcine model to study the effects of the sex hormone testosterone on scarring. Testosterone induces increased scar tissue deposition and appears to increase physical strength of scars via supraphysiologic deposition of collagen and other ECM factors. The increased burst strength seen in both XX and XY animals suggests that hormone administration has a strong influence on scar mechanical properties independent of chromosomal sex. Anti-androgen topical therapies may be a promising future area of research.

Oxidative stress, inflammation, blood rheology, and microcirculation in adults with sickle cell disease: Effects of hydroxyurea treatment and impact of sickle cell syndrome.

Inflammation and oxidative stress play a key role in the pathophysiology of sickle cell disease (SCD). However, the potential influence of different sickle genotypes, or hydroxyurea (HU) treatment, on these factors remains poorly documented. The present study compared several plasma markers of inflammation and oxidative stress, as well as microvascular function, between patients with sickle SC disease (HbSC, n = 19) and patients with sickle cell anemia (HbSS) under hydroxyurea (HU) treatment (n = 16), or not (n = 13). Hemorheological parameters and levels of inflammatory (IL-6, IL-8, IFN-γ, MCP-1, MIP-1ß, TNF-α) and oxidative stress (AOPP, MDA, MPO) markers were determined. Peripheral microcirculatory cutaneous blood flow and immediate microvascular response to local heat were evaluated using laser Doppler flowmetry. Oxidative stress and inflammation were lower in HbSC patients and HbSS patients under HU therapy compared to HbSS patients not treated with HU. Blood viscosity was higher in HbSC than in HbSS patients treated with or not with HU. Vasodilation response of the cutaneous microcirculation to heat stress was higher in HbSS patients receiving HU treatment. Our results clearly established that both sickle cell genotype and HU treatment modulate inflammation and oxidative stress.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

Klf4 has an unexpected protective role in perivascular cells within the microvasculature.

Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.

Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.

Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone.

OBJECTIVE: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. APPROACH AND RESULTS: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6(-/-) arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6(-/-) mice were protected against MT elevation in myocardial infarction-induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA-GTP binding, myosin light chain, P42-P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43(+/-) and P2rx7(-/-) mesenteric resistance arteries. CONCLUSIONS: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Regulation of cellular communication by signaling microdomains in the blood vessel wall.

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function.

Emerging concepts regarding pannexin 1 in the vasculature.

Pannexin channels are newly discovered ATP release channels expressed throughout the body. Pannexin 1 (Panx1) channels have become of great interest as they appear to participate in a multitude of signalling cascades, including regulation of vascular function. Although numerous Panx1 pharmacological inhibitors have been discovered, these inhibitors are not specific for Panx1 and have additional effects on other proteins. Therefore, molecular tools, such as RNA interference and knockout animals, are needed to demonstrate the role of pannexins in various cellular functions. This review focuses on the known roles of Panx1 related to purinergic signalling in the vasculature focusing on post-translational modifications and channel gating mechanisms that may participate in the regulated release of ATP.

Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction.

OBJECTIVE: Hemoglobin α (Hb α) and endothelial nitric oxide synthase (eNOS) form a macromolecular complex at myoendothelial junctions; the functional role of this interaction remains undefined. To test if coupling of eNOS and Hb α regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb α to disrupt this interaction. APPROACH AND RESULTS: In silico modeling of Hb α and eNOS identified a conserved sequence of interaction. By mutating portions of Hb α, we identified a specific sequence that binds eNOS. A mimetic peptide of the Hb α sequence (Hb α X) was generated to disrupt this complex. Using in vitro binding assays with purified Hb α and eNOS and ex vivo proximity ligation assays on resistance arteries, we have demonstrated that Hb α X significantly decreased interaction between eNOS and Hb α. Fluorescein isothiocyanate labeling of Hb α X revealed localization to holes in the internal elastic lamina (ie, myoendothelial junctions). To test the functional effects of Hb α X, we measured cyclic guanosine monophosphate and vascular reactivity. Our results reveal augmented cyclic guanosine monophosphate production and altered vasoconstriction with Hb α X. To test the in vivo effects of these peptides on blood pressure, normotensive and hypertensive mice were injected with Hb α X, which caused a significant decrease in blood pressure; injection of Hb α X into eNOS(-/-) mice had no effect. CONCLUSIONS: These results identify a novel sequence on Hb α that is important for Hb α/eNOS complex formation and is critical for nitric oxide signaling at myoendothelial junctions.

Loss of collectrin, an angiotensin-converting enzyme 2 homolog, uncouples endothelial nitric oxide synthase and causes hypertension and vascular dysfunction.

BACKGROUND: Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. METHODS AND RESULTS: Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates l-arginine uptake and plasma membrane levels of CAT1 and y(+)LAT1 amino acid transporters in endothelial cells. Treatment with l-arginine modestly lowers blood pressure of collectrin knockout mice. CONCLUSIONS: Collectrin is a consequential link between the transport of l-arginine and endothelial nitric oxide synthase uncoupling in hypertension.

The influence of gap junction network complexity on pulmonary artery smooth muscle reactivity in normoxic and chronically hypoxic conditions.

Experiments on intrapulmonary arteries (IPAs) isolated from rats maintained in normoxia and chronic hypobaric hypoxia showed that in normoxia, the IPA contractile sensitivity to KCl was not modified by gap junction inhibition. In contrast, chronic hypoxia induced an endothelium-independent hypersensitivity, which was suppressed by gap junction inhibition. For the theoretical analysis of these results, we developed a model of interconnected myocytes. Given that smooth muscle cells in IPAs are known to communicate via gap junctions, we regard the cytoarchitecture of the IPA as a spatial network, in which nodes represent individual smooth muscle cells and the links signify intercellular communication. A single-cell model that drives the dynamics of individual nodes includes the major elements of voltage-dependent Ca(2+) signalling. In addition, interindividual variability of SMCs is introduced by distributing the reversal potentials for K(+). Cell-to-cell connection consists of passive Ca(2+) diffusion and electrical coupling, and connection between cells is determined by the topology of the intercellular network. Model predictions indicate that the experimental results can be explained by topological modifications and not by changes in the number of gap junctions. According to the model, in normoxia the myocytes are connected in a complex network, whereas chronic hypoxia is related to loss of complexity, leading to hypersensitivity. Our results thus indicate that chronic hypoxia entails gap junction network rearrangements, leading to disturbances in the intercellular communication pathways.

Vascular biomechanical properties in mice with smooth muscle specific deletion of Ndst1.

Heparan sulfate proteoglycans act as co-receptors for many chemokines and growth factors. The sulfation pattern of the heparan sulfate chains is a critical regulatory step affecting the binding of chemokines and growth factors. N-deacetylase-N-sulfotransferase1 (Ndst1) is one of the first enzymes to catalyze sulfation. Previously published work has shown that HSPGs alter tangent moduli and stiffness of tissues and cells. We hypothesized that loss of Ndst1 in smooth muscle would lead to significant changes in heparan sulfate modification and the elastic properties of arteries. In line with this hypothesis, the axial tangent modulus was significantly decreased in aorta from mice lacking Ndst1 in smooth muscle (SM22αcre(+)Ndst1(-/-), p < 0.05, n = 5). The decrease in axial tangent modulus was associated with a significant switch in myosin and actin types and isoforms expressed in aorta and isolated aortic vascular smooth muscle cells. In contrast, no changes were found in the compliance of smaller thoracodorsal arteries of SM22αcre(+)Ndst1(-/-) mice. In summary, the major findings of this study were that targeted ablation of Ndst1 in smooth muscle cells results in altered biomechanical properties of aorta and differential expression of myosin and actin types and isoforms.

S-nitrosylation inhibits pannexin 1 channel function.

S-nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1-1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1(C40A), Panx1(C346A), and Panx1(C426A)). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1(C40A/C346A)) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.

Pannexin1 regulates α1-adrenergic receptor- mediated vasoconstriction.

RATIONALE: The coordination of vascular smooth muscle cell constriction plays an important role in vascular function, such as regulation of blood pressure; however, the mechanism responsible for vascular smooth muscle cell communication is not clear in the resistance vasculature. Pannexins (Panx) are purine-releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of vascular smooth muscle cell constriction. OBJECTIVE: We investigated the role of pannexins in phenylephrine- and KCl-mediated constriction of resistance arteries. METHODS AND RESULTS: Western blot, immunohistochemistry, and immunogold labeling coupled to scanning and transmission electron microscopy revealed the presence of Panx1 but not Panx2 or Panx3 in thoracodorsal resistance arteries. Functionally, the contractile response of pressurized thoracodorsal resistance arteries to phenylephrine was decreased significantly by multiple Panx inhibitors (mefloquine, probenecid, and (10)Panx1), ectonucleotidase (apyrase), and purinergic receptor inhibitors (suramin and reactive blue-2). Electroporation of thoracodorsal resistance arteries with either Panx1-green fluorescent protein or Panx1 small interfering RNA showed enhanced and decreased constriction, respectively, in response to phenylephrine. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with coimmunoprecipitation experiments from thoracodorsal resistance arteries, which suggested an association between Panx1 and α1D-adrenergic receptor. CONCLUSIONS: Our data demonstrate for the first time a key role for Panx1 in resistance arteries by contributing to the coordination of vascular smooth muscle cell constriction and possibly to the regulation of blood pressure.

Amount of Pannexin 1 in Smooth Muscle Cells Regulates Sympathetic Nerve-Induced Vasoconstriction.

BACKGROUND: Panx1 (pannexin 1) forms high conductance channels that secrete ATP upon stimulation. The role of Panx1 in mediating constriction in response to direct sympathetic nerve stimulation is not known. Additionally, it is unknown how the expression level of Panx1 in smooth muscle cells (SMCs) influences α-adrenergic responses. We hypothesized that the amount of Panx1 in SMCs dictates the levels of sympathetic constriction and blood pressure. METHODS: To test this hypothesis, we used genetically modified mouse models enabling expression of Panx1 in vascular cells to be varied. Electrical field stimulation on isolated arteries and blood pressure were assessed. RESULTS: Genetic deletion of SMC Panx1 prevented constriction by electric field stimulation of sympathetic nerves. Conversely, overexpression of Panx1 in SMCs using a ROSA26 transgenic model increased sympathetic nerve-mediated constriction. Connexin 43 hemichannel inhibitors did not alter constriction. Next, we evaluated the effects of altered SMC Panx1 expression on blood pressure. To do this, we created mice combining a global Panx1 deletion, with ROSA26-Panx1 under the control of an inducible SMC specific Cre (Myh11). This resulted in mice that could express only human Panx1, only in SMCs. After tamoxifen, these mice had increased blood pressure that was acutely decreased by the Panx1 inhibitor spironolactone. Control mice genetically devoid of Panx1 did not respond to spironolactone. CONCLUSIONS: These data suggest Panx1 in SMCs could regulate the extent of sympathetic nerve constriction and blood pressure. The results also show the feasibility humanized Panx1-mouse models to test pharmacological candidates.

Characterization of the thoracodorsal artery: morphology and reactivity.

OBJECTIVES: In this paper, we describe the histological and contractile properties of the thoracodorsal artery (TDA), which indirectly feeds the spinotrapezius muscle. METHODS: We used immunolabelling techniques to histologically characterize the TDA while the contractile properties were assessed using pressure arteriography. RESULTS: Our results demonstrate that the TDA is composed of approximately one to two layers of smooth muscle cells, is highly innervated with adrenergic nerves, and develops spontaneous tone at intraluminal pressures above 80 mmHg. The reactivity of the TDA in response to various contractile agonists such as phenylephrine, noradrenaline, angiotensin II, serotonin, endothelin 1, and ATP, as well as vasodilators, shows that the TDA exhibits a remarkably comparable reactivity to what has been observed in mesenteric arteries. We further studied the different components of the TDA response to acetylcholine, and found that the TDA was sensitive to TRAM 34, a blocker of the intermediate conductance potassium channel, which is highly suggestive of an endothelium-dependent hyperpolarization. CONCLUSIONS: We conclude that the TDA exhibits comparable characteristics to other current vascular models, with the additional advantage of being easily manipulated for molecular and ex vivo vasoreactivity studies.

Dehydroepiandrosterone reverses chronic hypoxia/reoxygenation-induced right ventricular dysfunction in rats.

Dehydroepiandrosterone (DHEA) prevents chronic hypoxia-induced pulmonary hypertension and associated right ventricle dysfunction in rats. In this animal model, reoxygenation following hypoxia reverses pulmonary hypertension but not right ventricle dysfunction. We thus studied the effect of DHEA on the right ventricle after reoxygenation, i.e. after a normoxic recovery phase secondary to chronic hypoxia in rats. Right ventricle function was assessed in vivo by Doppler echocardiography and in vitro by the isolated perfused heart technique in three groups of animals: control, recovery (21 days of hypoxia followed by 21 days of normoxia) and recovery DHEA (30 mg · kg(-1) every 2 days during the recovery phase). Right ventricle tissue was assessed by optical and electron microscopy. DHEA abolished right ventricle diastolic dysfunction, as the echographic E wave remained close to that of controls (mean ± SD 76.5 ± 2.4 and 79.7 ± 1.7 cm · s(-1), respectively), whereas it was diminished to 40.3 ± 3.7 in the recovery group. DHEA also abolished right ventricle systolic dysfunction, as shown by the inhibition of the increase in the slope of the pressure-volume curve in isolated heart. The DHEA effect was related to cardiac myocytes proliferation. In conclusion, DHEA prevents right ventricle dysfunction in this animal model by preventing cardiomyocyte alteration.

Posttranslational modifications in connexins and pannexins.

Posttranslational modification is a common cellular process that is used by cells to ensure a particular protein function. This can happen in a variety of ways, e.g., from the addition of phosphates or sugar residues to a particular amino acid, ensuring proper protein life cycle and function. In this review, we assess the evidence for ubiquitination, glycosylation, phosphorylation, S-nitrosylation as well as other modifications in connexins and pannexin proteins. Based on the literature, we find that posttranslational modifications are an important component of connexin and pannexin regulation.

Spontaneous lung dysfunction and fibrosis in mice lacking connexin 40 and endothelial cell connexin 43.

Gap junction proteins (connexins) facilitate intercellular communication and serve several roles in regulation of tissue function and remodeling. To examine the physiologic effects of depleting two prominent endothelial connexins, Cx40 and Cx43, transgenic mice were generated by breeding Cx40-deficient mice (Cx40(-/-)) with a vascular endothelial cell (VEC)-specific Cx43-deficient mouse strain (VEC Cx43(-/-)) to produce double-connexin knockout mice (VEC Cx43(-/-)/Cx40(-/-)). The life span in VEC Cx43(-/-)/Cx40(-/-) mice was dramatically shortened, which correlated with severe spontaneous lung abnormalities as the mice aged including increased fibrosis, aberrant alveolar remodeling, and increased lung fibroblast content. Moreover, VEC Cx43(-/-)/Cx40(-/-) mice exhibited cardiac hypertrophy and hypertension. Because VEC Cx43(-/-)/Cx40(-/-) mice demonstrated phenotypic hallmarks that were remarkably similar to those in mice deficient in caveolin-1, pulmonary caveolin expression was examined. Lungs from VEC Cx43(-/-)/Cx40(-/-) mice demonstrated significantly decreased expression of caveolin-1 and caveolin-2. This suggests that expression of caveolin-1 may be linked to expression of Cx40 and endothelial Cx43. Moreover, the phenotype of caveolin-1(-/-) mice and VEC Cx43(-/-)/Cx40(-/-) mice may arise via a common mechanism.